Stability studies of binding and functional anti-vaccine antibodies.

BACKGROUND: As most vaccines exert their protective capacity by eliciting pathogen-specific antibodies, antibody assays assessing immunogenicity of vaccines in development should be well characterized. Part of the validation of immunogenicity assays for vaccines is the study of stability of antibodies in serum. Materials & methods: Stability of antibodies in human serum was assessed by circumsporozoite-binding IgG ELISA designed for assessing the immunogenicity of a malaria vaccine under development, adenovirus neutralization assay, designed to assess neutralizing antibodies against adenovirus and commercially available test kits for hepatitis A and B. RESULTS: Stability studies indicated stability of serum-binding IgG antibodies and serum-neutralizing antibodies in: long-term storage below -65°C and -20°C; short-term storage; multiple freeze/thaw rounds; during shipment; and during heat inactivation. CONCLUSION: RESULTS have shown the stability of both binding and functional polyclonal antibodies in human serum under stable storage and common usage circumstances.

A peptide-based Plasmodium falciparum circumsporozoite assay to test for serum antibody responses to pre-erythrocyte malaria vaccines.

Various pre-erythrocyte malaria vaccines are currently in clinical development, and among these is the adenovirus serotype 35-based circumsporozoite (CS) vaccine produced on PER.C6 cells. Although the immunological correlate of protection against malaria remains to be established, the CS antibody titer is a good marker for evaluation of candidate vaccines. Here we describe the validation of an anti-Plasmodium falciparum circumsporozoite antibody enzyme-linked immunosorbent assay (ELISA) based on the binding of antibodies to a peptide antigen mimicking the CS repeat region. The interassay variability was determined to be below a coefficient of variation (CV) of 15%, and sensitivity was sufficient to detect low antibody titers in subjects from endemic regions. Antibody titers were in agreement with total antibody responses to the whole CS protein. Due to its simplicity and high performance, the ELISA is an easy and rapid method for assessment of pre-erythrocyte malaria vaccines based on CS.

Steric hindrance and fast dissociation explain the lack of immunogenicity of the minor histocompatibility HA-1Arg Null allele.

The di-allelic HLA-A2 restricted minor histocompatibility Ag HA-1 locus codes for the highly immunogenic HA-1(His) and the nonimmunogenic HA-1(Arg) nonapeptides, differing in one amino acid. The HA-1(His) peptide is currently used for boosting the graft-vs-tumor responses after HLA matched HA-1 mismatched stem cell transplantation; usage of the HA-1(Arg) peptide would significantly enlarge the applicability for this therapy. Our studies on mechanisms causing the HA-1 unidirectional immunogenicity revealed marginal differences in proteasomal digestion, TAP translocation, and binding affinity, whereas both dissociation rates and structural analyses clearly showed marked differences in the stability of these two HLA-A2 bound alleles. These data provide a rationale for the lack of HA-1(Arg) peptide immunogenicity essential for the choice of tumor peptides for stem cell-based immunotherapeutic application.

Adult and cord blood T cells can acquire HA-1 specificity through HA-1 T-cell receptor gene transfer.

BACKGROUND AND OBJECTIVES: Minor histocompatibility antigen (mHag)-specific graft-versus-leukemia reactivities are observed following unselected donor lymphocyte infusion for the treatment of relapse after HLA-matched mHag-mismatched stem cell transplantation (SCT). Adoptive transfer of donor-derived ex vivo-generated HA-1-specific oligoclonal T cells or HA-1 peptide patient vaccination are currently being explored as curative tools for stem cell based immunotherapy of hematologic malignancies. Another treatment modality to eradicate residual leukemic cells after SCT is the transfer of the HA-1 hematopoietic-specific T-cell receptor (TCR) into cells from the stem cell donor. This strategy would be particularly useful in case of relapse after cord blood transplantation (CBT) and is explored in this study. DESIGN AND METHODS: HLA-A2(neg) adult peripheral blood and cord blood mononuclear cells were transduced with the genes encoding the HA-1 alpha and beta TCR chains derived from established HA-1 specific cytotoxic T lymphocyte clones. RESULTS: The T cells transduced with HA-1 TCR alpha beta showed consistent marker gene expression, but low staining with HLA-A2/HA-1 tetrameric complexes. They did, however, show hematopoietic-restricted cytolytic activity against HLA-A2(pos)/HA-1(pos) target cells, including leukemic cells. INTERPRETATION AND CONCLUSIONS: The low level of HA-1-specific tetramer staining of HA-1 TCR alpha beta transduced T cells may be caused by hybrid TCR formation of the transferred TCRalpha and beta chains with endogenous TCR alpha and beta chains. This may cause unwanted alloreactivity and requires attention. The HA-1 TCR alpha beta transduced T cells show that the HA-1 TCR can be functionally transferred into donor mononuclear cells, which can be exploited in immunotherapeutic settings of SCT and CBT for hematologic malignancies.

Cord blood comprises antigen-experienced T cells specific for maternal minor histocompatibility antigen HA-1.

Umbilical cord blood transplantation is applied as treatment for mainly pediatric patients with hematologic malignancies. The clinical results show a relatively low incidence of graft-versus-host disease and leukemia relapse. Since maternal cells traffic into the fetus during pregnancy, we questioned whether cord blood has the potential to generate cytotoxic T cells specific for the hematopoietic minor histocompatibility (H) antigen HA-1 that would support the graft-versus-leukemia effect. Here, we demonstrate the feasibility of ex vivo generation of minor H antigen HA-1-specific T cells from cord blood cells. Moreover, we observed pre-existing HA-1-specific T cells in cord blood samples. Both the circulating and the ex vivo-generated HA-1-specific T cells show specific and hematopoietic restricted lysis of human leukocyte antigen-A2(pos)/HA-1(pos) (HLA-A2(pos)/HA-1(pos)) target cells, including leukemic cells. The cord blood-derived HA-1-specific cytotoxic T cells are from child origin. Thus, the so-called naive cord blood can comprise cytotoxic T cells directed at the maternal minor H antigen HA-1. The apparent immunization status of cord blood may well contribute to the in vivo graft-versus-leukemia activity after transplantation. Moreover, since the fetus cannot be primed against Y chromosome-encoded minor H antigens, cord blood is an attractive stem cell source for male patients.

Competition-based cellular peptide binding assays for 13 prevalent HLA class I alleles using fluorescein-labeled synthetic peptides.

We report the development, validation, and application of competition-based peptide binding assays for 13 prevalent human leukocyte antigen (HLA) class I alleles. The assays are based on peptide binding to HLA molecules on living cells carrying the particular allele. Competition for binding between the test peptide of interest and a fluorescein-labeled HLA class I binding peptide is used as read out. The use of cell membrane-bound HLA class I molecules circumvents the need for laborious biochemical purification of these molecules in soluble form. Previously, we have applied this principle for HLA-A2 and HLA-A3. We now describe the assays for HLA-A1, HLA-A11, HLA-A24, HLA-A68, HLA-B7, HLA-B8, HLA-B14, HLA-B35, HLA-B60, HLA-B61, and HLA-B62. Together with HLA-A2 and HLA-A3, these alleles cover more than 95% of the Caucasian population. Several allele-specific parameters were determined for each assay. Using these assays, we identified novel HLA class I high-affinity binding peptides from HIVpol, p53, PRAME, and minor histocompatibility antigen HA-1. Thus these convenient and accurate peptide-binding assays will be useful for the identification of putative cytotoxic T lymphocyte epitopes presented on a diverse array of HLA class I molecules.

Identification of a novel HLA-B60-restricted T cell epitope of the minor histocompatibility antigen HA-1 locus.

The polymorphic minor histocompatibility Ag HA-1 locus encodes two peptides, HA-1(H) and HA-1(R), with a single amino acid difference. Whereas the immunogenicity of the HA-1(R) allele has not yet been shown, the nonameric HA-1(H) peptide induces HLA-A2-restricted cytotoxic T cells in vivo and in vitro. It is not known whether the mHag HA-1(H) or HA-1(R) associates with other HLA class I molecules. Therefore, the polymorphic regions of both HA-1 alleles were analyzed to identify HLA class I binding peptides that are properly processed by proteasomal degradation. Peptide binding analyses were performed for all nonameric HA-1(H/R) peptides for binding to nine HLA class I molecules with >10% prevalence in the Caucasian population and for seven nonameric/decameric HA-1(H/R) peptides predicted to bind to HLA-A3, -B14, and -B60. Only the nonameric KECVL(H)/(R)DDL and decameric KECVL(H)/(R)DDLL peptides showed strong and stable binding to HLA-B60. In vitro digestion of 29-aa-long HA-1 peptides by purified 20S proteasomes revealed proper cleavage at the COOH termini of both HLA-B60 binding HA-1(H) and HA-1(R) peptides. In subsequent analyses, dendritic cells pulsed with the nonameric HA-1(R) peptide did not induce CTLs that recognize the natural HLA-B60/HA-1(R) ligand. In contrast, dendritic cells pulsed with the nonameric HA-1(H) peptide induced IFN-gamma-secreting T cells specific for the natural HLA-B60/HA-1(H) ligand in three HLA-B60(+) HA-1(RR) individuals, demonstrating the immunogenicity of the HLA-B60/HA-1(H) ligand. In conclusion, this study shows a novel HLA-B60-restricted T cell epitope of the minor histocompatibility Ag HA-1 locus.